Journal of Inorganic Biochemistry 104 (2010) 726–731

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Journal of Inorganic Biochemistry

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Metal-dependent inhibition of glyoxalase II: A possible mechanism to regulate theenzyme activity

Valeria A. Campos-Bermudez a,1, Jorgelina Morán-Barrio a, Antonio J. Costa-Filho b, Alejandro J. Vila a,⁎a IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas,Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentinab Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil

⁎ Corresponding author. Fax: +54 341 4390465.E-mail addresses: campos@cefobi-conicet.gov.ar (V.A

moran@ibr.gov.ar (J. Morán-Barrio), ajcosta@if.sc.usp.brvila@ibr.gov.ar (A.J. Vila).

1 Present address: CEFOBI (Centro de Estudios FotosinNacional de Investigaciones Científicas y Técnicas (COBioquímicas y Farmacéuticas, UniversidadNacional deRosRosario, Argentina.

0162-0134/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.jinorgbio.2010.03.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 August 2009Received in revised form 15 February 2010Accepted 9 March 2010Available online 20 March 2010

Keywords:Glyoxalase IIIronEPRProduct inhibitionParamagnetic NMR

Glyoxalase II (GLX2, EC 3.1.2.6., hydroxyacylglutathione hydrolase) is a metalloenzyme involved in crucialdetoxification pathways. Different studies have failed in identifying the native metal ion of this enzyme,which is expressed with iron, zinc and/or manganese. Here we report that GloB, the GLX2 from Salmonellatyphimurium, is differentially inhibited by glutathione (a reaction product) depending on the bound metalion, and we provide a structural model for this inhibition mode. This metal-dependent inhibition was shownto occur in metal-enriched forms of the enzyme, complementing the spectroscopic data. Based on the highlevels of free glutathione in the cell, we suggest that the expression of the different metal forms of GLX2during Salmonella infection could be exploited as a mechanism to regulate the enzyme activity.

. Campos-Bermudez),(A.J. Costa-Filho),

téticos y Bioquímicos), ConsejoNICET), Facultad de Cienciasario, Suipacha 531, (S2002LRK),

l rights reserved.

© 2010 Elsevier Inc. All rights reserved.

1. Introduction

The ubiquitous glyoxalase system is composed of two metalloen-zymes (glyoxalase I and glyoxalase II), whose physiological role is tocatalyze the conversion of methylglyoxal to the corresponding 2-hydroxycarboxylic acids [1]. Glyoxalase I (GLX1, EC 4.4.1.5., lactoylglu-tathione methylglyoxal lyase) converts a thiohemiacetal (formed frommethylglyoxal and glutathione) to S-D-lactoylglutathione (SLG), whileglyoxalase II (GLX2) takes the latter as a substrate and catalyzes itshydrolysis to yield D-lactic acid and glutathione (GSH) [2,3] (Fig. 1A).

Although GLX2 is able to hydrolyze many different glutathionethioesters, S-D-lactoylglutathione (SLG) is the preferred substrate forthis enzyme from most sources including human, yeast, and plants.The glyoxalase enzymatic system is essential for chemical detoxifica-tion, and therefore has been identified as a target for the developmentof anti-cancer and anti-protozoan drugs [4–6].

GLX2 belongs to the metallo-β-lactamase superfamily, which ischaracterized by an αβ/βα fold containing a conserved motif able tobind up to two metal ions in their active sites. Most hydrolases fromthis superfamily use ZnII, a redox inactivemetal ion for their roles, such

as metallo-β-lactamases [7,8], N-acyl homoserine (AHL) lactonases[9,10] and tRNAseZ [11], whereas the terminal oxidase rubredoxin:oxygen oxidoreductase (ROO) from D. gigas contains a binuclear ironsite [12–14]. GLX2, despite exhibiting the same coordination spheredisplayed by other hydrolases from this superfamily (Fig. 1B), has beenan outstanding exception regarding the metal ion dependence withdifferent metal cofactors [15–20].

In general, the wild type forms of the recombinant GLX2 isoen-zymes characterized up to nowwere isolated with varying amounts ofiron, zinc and manganese bound to their active sites [15–20]. Sur-prisingly, the different metal derivatives show comparable high cata-lytic efficiencies [15–20]. An exception to this observation is humanGLX2, which has been reported to be inactive in its Mn-bound form[20]. Despite the existence of three crystallographic structures (2QED,1QH5, 1XM8) [18,19,21] which have been refinedwith differentmetalions in its active sites, this puzzling promiscuity has precluded theidentification of the native metal ion in GLX2.

Most mechanistic proposals so far have considered exclusively thedi-ZnII derivative, based on crystallographic, kinetic and spectroscopicstudies, and (more recently) theoretical calculations at the quantumlevel [17,21,22] (Fig. 1B). However, onemay arguewhether thismech-anism could be general to all metal derivatives.

Here we report a spectroscopic and kinetic study on GloB, theglyoxalase II from Salmonella typhimurium [19], upon addition ofsubstrate S-D-lactoylglutathione (SLG) or the product glutathione(GSH). We have interrogated the enzyme purified from rich medium,containingmixedmetallic centers, aswell as Fe-, Zn- andMn-enrichedforms [19]. We have found that GloB is differentially inhibited by

Fig. 1. (A) Reaction catalyzed by glyoxalase II (GLX2). (B) Proposed reactionmechanism for dimetallic-GLX2 based on crystallographic, kinetic and theoretical calculations. Themetalion bound to 3 His residues (M1) is proposed to deliver the attacking nucleophile, while the M2 site favors S–C bond cleavage by stabilizing the negative charge in the sulfur atom ofthe glutathione moiety.

727V.A. Campos-Bermudez et al. / Journal of Inorganic Biochemistry 104 (2010) 726–731

glutathione depending on the bound metal ion, and we provide astructural model for this inhibition mode.

2. Experimental procedure

2.1. General

S-D-lactoylglutathione (SLG) was purchased from Sigma-Aldrich.All chromatographic steps were performed in an Amersham Bios-ciences liquid chromatography system operating at 4 °C. Metal stan-dards were purchased from Fisher Scientific and were diluted withdistilled water. All other chemicals used in this study were purchasedcommercially and were of the highest quality available.

2.2. Protein overexpression and purification

GLX2 (GloB) from S. typhimurium 14028s recombinantly producedin E. coli, was used for this study. Protein overexpression and purifi-cation were performed using the pET32-gloB vector, as previouslydescribed [19]. Fractions with GLX2 activity were pooled and dialyzedagainst 10 mM4-morpholinepropanesulfonic acid (MOPS) 0.2 MNaClat pH 7.2. In order to selectively produce metal-enriched enzyme

forms, minimal media M9 was employed in culture growth supple-mented with metal ions from stock solutions of Fe(NH4)SO4, MnCl2,or ZnCl2, to reach a final concentration of 100 μM in the culture.The minimal media contained 4 g/L D-(+)-glucose (Sigma), 12.8 g/LNa2HPO4·7H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1.0 g/L ammonium sul-fate, 10 μM CaCl2 and 1 mMMgSO4. The metal ions were added in themoment of induction, when the growth cultures reached an OD600 of0.6–0.8. The purification protocol is described in Ref. [19]. Thus, weobtained the iron-enriched GloB (FeGloB), manganese-enriched GloB(MnGloB) and zinc-enriched GloB (ZnGloB).

2.3. Protein concentration

Enzyme concentration was determined by measuring the A280nm

and using the extinction coefficient ε280nm=28,030 M−1 cm−1 [19].This parameter was calculated from amino acid composition infor-mation by applying a modified Edelhoch method [23].

2.4. Metal analysis

Themetal content of the GloB samples was measured using atomicabsorption spectroscopy in a Metrolab 250 AA instrument. Purified

Fig. 2. Experimental (upper traces) and calculated (lower traces) X-band EPR spectra of1 mM GloB (A) as isolated; (B) after the addition of SLG and (C) after the addition ofGSH. The spectra were recorded at 4.7 K and simulated as described in the text. Thearrows indicate approximate g-values.

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enzymes were diluted with 10 mM MOPS pH 7.2, to a concentrationof 30 μM and analyzed for zinc, manganese and iron. The metalcontent of GloB produced in minimal medium was the following:FeGloB: 0.86±0.02 eq iron, 0.05±0.02 eq manganese and 0.04±0.01 eq zinc; MnGloB: 0.04±0.01 eq iron, 0.81±0.06 eq manganeseand 0.04±0.01 eq zinc; ZnGloB: 0.08±0.01 eq iron, 1.42±0.01 eqzinc and manganese was not detected. The metal content data pre-sented in this paper represent an average from at least three pre-parations of each growth condition.

2.5. 1H NMR spectroscopy

NMR spectra were recorded in a Bruker Avance II 600 spectrom-eter operating at 600.13 MHz. 1H NMR spectra were recorded underconditions to optimize detection of the fast-relaxing paramagneticresonances, either using the superWEFT pulse sequence [24,25] orwater presaturation. Spectra were acquired over large spectral widthswith acquisition times ranging from 16 to 80 ms, and intermediatedelays from 2 to 35 ms. 1D experiments with solvent presaturationwere used to record isotropically shifted signals closer to the dia-magnetic envelope. The samples were loaded into Wilmad 5-mmNMR tubes. The substrate or product was prepared as 100 mM stocksolutions in 100 mM MOPS at pH 7.2 (pD 6.8). A final 1:10 enzymeto reactant ratio was used to record the spectra in presence of theadducts.

2.6. Electron paramagnetic resonance spectroscopy

X-band (9.5 GHz) EPR spectraweremeasured on a Bruker ELEXSYSE580 system (Bruker BioSpin, Germany) at 4.7 K. The temperaturewas controlled with an Oxford ITC503 cryogenic system. EPR samplescontaining a convenient amount of protein or the protein to whichthe substrate or product were added (up to a 1:10 ratio) were frozenby immersion in liquid nitrogen and then placed in the spectro-meter rectangular cavity. All EPR data were corrected by subtractinga baseline corresponding to the EPR signal of the buffer. Other acqui-sition conditions: modulation amplitude, 1 mT; modulation frequen-cy, 100 kHz;microwave power, 4 mW.A typical EPR samplewas 1 mMin 10 mM MOPS pH 7.2 buffer.

2.7. UV–visible spectroscopy

Spectra of GloB samples in the absence and presence of substrateor product in different ratios, were recorded between 240 and 800 nmat 25 °C in a Jasco 550 UV–vis spectrophotometer in quartz cuvette of1 cm of path length. Differential spectra were obtained by subtractingthe spectrum of the enzyme in its unbound form. Typical samples ofGloB containing different metal ions were up to 1 mM in protein,while the spectra of Fe-enriched, Mn-enriched and Zn-enriched wererecorded on samples typically ca. 30 μM.

2.8. End product inhibition studies of SLG hydrolysis

Inhibition studies were performed on selectively metal-enrichedGloB using glutathione, one of the products of SLG hydrolysis. Steadystate kinetic studies of purified GloB variants in the presence of vary-ing concentrations of GSH (0.2 to 5 mM) were conducted by mea-suring the rate of hydrolysis of a fixed concentration of SLG (400 μM)at 240 nm (ε240=3100 M−1 cm−1). A glutathione stock solutionwas prepared in 100 mM MOPS, pH 7.2. The enzyme (400 nM) andinhibitor were incubated, and then the substrate was added. IC50

values were determined by fitting the data of initial rates to thefollowing equation: (1−vi /vo)⁎100=Imax.[I] /(IC50+[I]). Measure-ments were performed at least by triplicate in a reaction volumeof 1 ml of 10 mM MOPS, 0.2 M NaCl pH 7.2 buffer, at 30 °C in a Jasco550 UV–vis spectrophotometer.

3. Results

GloB was obtained as previously described in Campos-Bermudezet al. [19]. The typical metal content of enzyme samples expressed inrich medium is 0.2 zinc; 0.6 iron and 0.3 manganese per protein.Selectively metallated variants were produced by expressing the pro-tein in minimal medium adequately supplemented with the requiredmetal ion. The behavior of these proteins upon the addition of sub-strate or product was analyzed by different spectroscopic techniques.

3.1. Electron paramagnetic resonance

The EPR spectrum of resting-state GloB recorded at 4.7 K (Fig. 2A)disclosed the coexistence of different paramagnetic species. As alreadyshown, the different observed signals can be assigned on the basis ofspectral simulations [19]. Themain components in the EPR spectra aredue to two different iron centers. Resonances at geff∼4.3 and 9.1 arecharacteristics of magnetically isolated high-spin FeIII in a rhombicenvironment [15,18,19,26–28]. The broad feature at gb2 correspondsto an antiferromagnetically coupled dinuclear center which involvesa high-spin (S=5/2) FeIII ion and a high-spin (S=2) FeII ion [15,18,19,28]. The six-line pattern centered around g=2.0 is due to MnII

bound to the active site (Fig. 2A).S-D-lactoylglutathione (SLG, the enzyme substrate) was added to

GloB, and the EPR spectrum was recorded after manual mixing andsample freezing. The spectrum (Fig. 2B) revealed changes in the reso-nances stemming from the iron centers at geff∼4.3, 9.1 and b2, whilethe signals arising from the MnII site could be accounted for in thesimulation by the same set of parameters obtained for resting-stateGloB, suggesting that the MnII ions are not perturbed. Addition ofglutathione (GSH, a reaction product), gave rise to similar changes onthe EPR signals corresponding to the iron centers, while those fromthe MnII sites also remained unperturbed (Fig. 2C).

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Analysis of the individual features revealed that in the presence ofSLG or GSH, the uncoupled signal (geff∼4.3) presented higher zero-field splittings (D=0.55 cm−1 and E/D=0.285) compared to resting-state GloB (D=0.33 cm−1 and E/D=0.195). The increase in D-valuescan be attributed to a change in the coordination sphere of the ironion, with more electron-donating atoms; while the higher E/D ratioreveals a larger rhombicity in the FeIII center. Both parameters areconsistent with the binding of an additional ligand to the FeIII ion[29,30].

Regarding the signal accounting for the coupled center (gb2), theaddition of either SLG or GSH led to lineshape and g-value differen-ces (Fig. 2B–C), revealing that the environment of the binuclear centeris significantly perturbed in both cases. Spectra of coupled centersare very sensitive to changes in the coordination environment of thebinuclear site which can be reflected in the magnitude of the super-exchange coupling (J) between the metal centers, and the zero-fieldsplittings (D) of each ion, among other features [31–34]. Effectiveg-values for these systems could be easily determined assuming anS=1/2 spin system and yielded, in the presence of ligands, 1.95, 1.87,and 1.71 (gav=1.84). Thus, the addition of exogenous ligands (SLGor GSH) is reflected as a significant increase in gav value for GloB inthe presence of ligands (gav=1.84) as compared to pure GloB (gav=1.76), which suggests a stronger coupling regime upon ligand binding.

The EPR spectra of Fe-enriched GloB before and after addition ofsubstrate (SLG) or product (GSH) revealed the same spectral featuresthat for the heterogeneous sample of GloB (not shown). The spectradisclosed mononuclear and binuclear Fe centers, and a weak six-linepattern. Double-integration of the spectra showed that this six-linepattern is responsible for less than 5% of the total signal intensity,indicating that this is the result of a very minor contribution from Mncenters in the sample, which is in agreement with metal analyses. Thespectra showed that addition of substrate (SLG) or product (GSH), ledto the same spectral alterations that those observed in Fig. 2B–C.

3.2. 1H nuclear magnetic resonance

The 1H NMR spectrum of GloB was recorded under conditionstailored to optimize detection of the fast-relaxing signals close to theparamagnetic metal center [25]. The spectrum of GloB reveals a setof hyperfine-shifted signals attributed to the His and Asp ligands ofthe metal site (Fig. 3) of a weakly antiferromagnetic coupled FeIII/FeII

Fig. 3. 1H NMR spectra of GloB (A) as isolated, (B) after the addition of SLG and (C) afterthe addition of GSH. The spectra were recorded at 600 MHz, pH 7.2, and 298 K in 10 mMMOPS buffer, 0.2 M NaCl.

system. Resonances corresponding to an uncoupled FeIII center can-not be detected, since considerably broader lines are expected [35].The addition of excess substrate (SLG) to GloB, resulted in a new setof resonances sharper than those from the resting-state enzyme(Fig. 3B). Addition of GSH to GloB rendered a spectrum very similar tothat resulting upon substrate addition (Fig. 3C). Since NMR spectra arerecorded at room temperature, in contrast with EPR, and NMR acqui-sition times are rather long (in relation with the GloB kcat=170 s−1),the time stability of the monitored species suggests that all studiedspectra correspond to an enzyme–product adduct. We must recallthat the glutathione moiety is present both in the substrate and pro-duct molecules (Fig. 1A).

3.3. UV–vis spectroscopy

To test the hypothesis that the formed species is an enzyme–product complex, the interaction of GloB with SLG and GSH was fol-lowed by UV–vis spectroscopy. We initially attempted these experi-ments with a sample of GloB obtained from expression in rich media,containing Fe, Zn and Mn. The addition of excess SLG resulted inthe appearance of a blue color, corresponding to an absorption bandcentered at 590 nm, with an estimated extinction coefficient of300 M−1 cm−1 (Fig. 4A). This spectral feature vanished after severalhours, confirming the formation of a stable adduct with the enzyme.This feature is in good agreement with the presence of a ligand-to-metal charge transfer (LMCT) band of a Cys ligand to a high-spin FeIII

in an octahedral coordination, reminiscent of those observed in super-oxide reductase and model complexes [36–38]. An additional intenseband at 320 nm is observed in the differential spectrum (Fig. 4A),which can be assigned to a (Cys)S–FeII LMCT [38]. Similar UV–visspecta were obtained for Tflp, a ferredoxin-like protein from Thermo-anaerobacter tengcongensis [39] belonging to the metallo-β-lactamasesuperfamily, where a disulfide bond is near the di-Fe center in theactive site.

When GSH was added to GloB, the same spectral features wereobserved (Fig. 4A). These results further confirm that the spectra

Fig. 4. Differential UV–visible spectrum of GloB produced in rich medium after theaddition of substrate or product (A) and from minimal medium supplemented eitherwith FeII (FeGloB), MnII (MnGloB) or ZnII (ZnGloB) after the addition of 10 equivalentsof substrate SLG (B). The differential spectra were obtained by subtracting the spectrumof unbound GloB. The observed features are in good agreement with the presence of aligand-to-metal charge transfer (LMCT) band of a Cys ligand to iron ions.

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recorded after addition of SLG correspond to a stable enzyme–product(E–P) adduct, formed as the result of the interaction of the metal sitewith the Cys-containing glutathione moiety of the substrate (Fig. 1A).These data are consistent with the crystal structure of the humanenzyme complexed with GSH (1QH5), in which the sulfur atom of thisCys residue is at 2.86 Å from one of themetal ions [21]. The absorptionfeature at 590 nm observed for the GloB–GSH complex displays arelatively low extinction coefficient, which is consistent with therelatively long S–FeIII bond reported in the crystal structure. The lackof a spectral feature at 280 nm allows us to discard the presence of aCys–MnII LMCT, i.e., confirming that this adduct is exclusively formedby the iron form of GLX2 [36].

When the same experiments were performed in the Fe-enrichedform, obtained by expression in minimal medium supplemented withFeII, we observed the same LMCT bands upon addition of either SLGor GSH. These experiments unequivocally confirm that the featuresobserved in the mixed GloB sample correspond to the iron form.Instead, when similar titrations were conducted in the Mn-enrichedand Zn-enriched forms of GloB, no distinctive features could be de-tected in the differential spectra even upon addition of a 10-foldexcess of the exogenous ligands up to 240 nm (Fig. 4B).Minor changesin the spectra can be attributed to alterations in the absorption fea-tures of the aromatic residues, as results from the fine structure ofthe differential spectra. These data allow us to discard the formationof a Cys–MnII CT band, which is expected to appear at 280 nm. This isvalidated by comparison with the spectrum obtained with the Znform of the enzyme, which it is not expected to exhibit a LMCT in thisspectral range.

3.4. Product inhibition of the specific metallated forms

The differential behavior of the different metallated forms towardsthe formation of enzyme–product complexes led us to analyze pro-duct inhibition of each metal form of GloB. We obtained the Mn-enriched, Zn-enriched and Fe-enriched forms of GloB, by expressionin minimal medium as already described [19], and we tested theirinhibition by glutathione. Fe- and Zn-GloB were inhibited at submil-limolar concentrations of glutathione, while the IC50 value for Mn-GloB is 1.5 mM. These results are in agreement with the spectrosco-pic data, which show strong product binding to the iron-substitutedenzyme, and not to the manganese forms. These data also reveal thatthe behavior of the Zn form resembles that of Fe-GloB.

4. Discussion

Glyoxalase II belongs to themetallo-β-lactamase superfamily, whichincludes several zinc-dependent hydrolases, such as metallo-β-lacta-mases, endonuclease tRNase Z, AHL lactonase and phosphorylcholineesterase [40]. GLX2 is an exception in that different metal derivativesshow sizably high catalytic performances for most isoforms [15–20].This is valid for the plant and bacterial isozymes characterized so far,since, despite high sequence homology, human GLX2 is inactive inits MnII form [20]. This fact suggests that metal discrimination couldplay a role in glyoxalase activity.

The proposed mechanism for the di-Zn enzyme assumes bindingof S-D-lactoylglutathione substrate to the enzyme, mainly by recog-nition of the glutathione moiety by active site residues, excludinga direct interaction with the metal ion (Fig. 1B) [22]. The metal ionbound to 3 His residues (M1) is proposed to deliver the attackingnucleophile, at the same time stabilizing the development of negativecharge in an oxygen atom during formation of a tetrahedral inter-mediate. After the nucleophilic attack, the M2 site favors S–C bondcleavage by stabilizing the negative charge in the sulfur atom ofthe glutathione moiety. After protonation and bond cleavage, a lac-tate and a glutathione moiety remains bound to the M1 and M2 ions,

respectively (Fig. 1B). A sequential mechanism for product release hasbeen proposed [17,21,22].

Here we report a metal-selective product inhibition, which allowus to obtain somemechanistic insights. By using complementary spec-troscopic techniques, we provide evidence supporting that: (1) addi-tion of either substrate (SLG) or product (GSH) to GloB results in theformation of a stable enzyme–product (GloB–GSH) complex; (2) thisadduct is formed by the iron variants of the enzyme, and not by themanganese derivatives; (3) a thiol–Fe bond is formed between theCys moiety of GSH and the metal site; and (4) the dinuclear ironsites show an enhanced antiferromagnetic coupling in the GloB–GSHadduct.

The finding of a stable thiol–Fe bonding interaction can be readilyattributed to product inhibition by glutathione, supporting a sequen-tial, ordered mechanism for product release after hydrolysis [17,21,22]. The reported lower KI for glutathione suggests that it is boundmore tightly to the active site and therefore is released after D-lacticacid [17].

EPR spectroscopy reveals that the coupling between the two ironions is stronger in the enzyme–product complex. This coupling is ex-pected to occur by involving Asp127 as a bridging ligand (Fig. 5). Onepossible rationale for this observation is to assume an equilibriumbetween the bridged and non-bridged forms in the free enzyme,whichis consistent with the variable geometries observed in different crystalstructures [15,18,19,21], that would be shifted towards the bridgedone upon formation of the EP adduct (Fig. 5A). Sharing the Asp127ligand between the two metal ions upon thiolate binding may com-pensate the increase in negative charge in the metal coordinationsphere. Another possibility is that the sulfur atom from glutathioneforms a strong hydrogen bond to the bridging hydroxide, thus impart-ing more oxo character in the bridging unit, giving rise to a strongercoupling.

Finally, we could also assume that the thiolate group of glutathi-one is bound to the metal site bridging the two iron ions (Fig. 5B),in a position equivalent to the one occupied by the nucleophile inthe resting-state enzyme. The GloB–GSH complex displays S–thiol–FeIII and S–thiol–FeII: CT bands (Fig. 4). This observation could be ac-counted for by assuming a bridging glutathione moiety, confirmingagain that product release is ordered in a sequential fashion.

The most relevant observation regarding this enzyme–productcomplex concerns its metal selectivity. MnII–thiolate bonds are lesscovalent and weaker than iron–thiolate bonds, providing a rationalefor this differential behavior [36]. This is also consistent with the find-ing that, in general, the MnII forms of different GLX2s are the mostefficient ones [15,16,19,41].

FeII and MnII are regarded as the most important transition metalions involved in host–bacterial pathogen interactions [43]. The acqui-sitions of both metal ions, and particularly MnII, are required for in-tracellular survival and replication of Salmonella enterica serovartyphimurium in macrophages in vitro and for virulence in vivo [44,45].Thus, fine regulation of metal ions availability in vivo could determinethe pathogen survival inside host cells. Since the levels of free gluta-thione in vivo are high, this differential inhibition mode might betaken as an indication that MnII is the nativemetal ion, in line with ourresults. However, additional experimental work is needed to statethat.

The glyoxalase system in Salmonella strains is the main defensemechanism to the accumulation of methylglyoxal generated in thephagolysosome during infection [42]. In contrast to other genesinvolved in methylglyoxal detoxification, that are induced in theSalmonella-containing vacuole during infection (such as those codingGLX1), the expression levels of gloB are constant through the patho-gen infection cycle [42]. Based on the evidence herein presented thatshows that inhibition by GSH is metal-dependent, the assembly of aspecific metal-enriched form of GLX2 could be exploited as a mech-anism to regulate the enzyme activity in vivo.

Fig. 5. Cartoon of the active site of GloB during catalysis. The equilibrium between a bridging Asp (left, top) and non-bridging Asp (left, bottom) in the free enzyme is shifted towardsthe bridged one in the GSH-bound form (right) (A). The thiolate group of glutathione bound to the metal site bridging the two iron ions upon formation of the EP adduct (B).

731V.A. Campos-Bermudez et al. / Journal of Inorganic Biochemistry 104 (2010) 726–731

AbbreviationsGLX glyoxalaseEPR electron paramagnetic resonanceRMN nuclear magnetic resonanceSLG S-D-lactoylglutathioneGSH glutathioneE–P enzyme–product adduct

Acknowledgements

This work was supported by grants from ANPCyT and HHMI to AJVand from PRONEX/FAPESP/CNPq (Grant no 03/09859-2) and CNPq(307102/2006-8) to AJCF. VCB and JMB are recipients of fellowshipsfrom CONICET. AJV is a Staff member from CONICET and an Inter-national Research Scholar of theHowardHughesMedical Institute. The600 MHz NMR spectrometer was purchased with funds from ANPCyT(PME2003-0026) and CONICET.

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